Published May 1, 1986
Subaxolemmal Cytoskeleton in Squid Giant Axon.
I. Biochemical Analysis of Microtubules, Microfilaments,
and Their Associated High-Molecular-Weight Proteins
T a k a a k i Kobayashi,* Shoichiro Tsukita,* S a c h i k o Tsukita,* Y o s h i o Y a m a m o t o , ~ and
Gen Matsumoto ~
*Department of Biochemistry, Jikei University, School of Medicine, Minato-ku, Tokyo 105; *Department of Anatomy,
Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo I 13; *Electrotechnical Laboratory, Tsukuba Science City,
Ibaraki 305, Japan. The present address of Drs. Shoichiro Tsukita and Sachiko Tsukita is Ultrastructural Research Section,
Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
Abstract. Using the squid giant axon, we analyzed
was soluble in 0.6 M NaC1 solution but insoluble in
0.1 M NaC1 solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin
molecule in 0.6 M NaCI solution looked like a straight
rod ~105 nm in length with a globular head at one
end. On the other hand, the purified 255-kD protein
was soluble in both 0.1 and 0.6 M NaC1 solution and
co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as
a characteristic horseshoe-shaped structure ~35 nm in
diameter. Furthermore, the 255-kD protein showed
no cross-reactivity to the anti-axolinin antibody.
Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid
giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated
H M W protein (axolinin), and actin filaments and an
actin filament-associated H M W protein (255-kD protein).
VIDENCEhas accumulated that the cytoskeletal network
underlying the plasmalemma plays an important role
in regulating the activities of the membrane in various
types of cells. In this respect, the physiological roles ofaxonal
cytoskeleton in the excitation process of the axolemma appear
to be of interest. To analyze this problem, correlative physiological, biochemical, and morphological studies are required,
and the squid giant axon offers an advantageous model for
this purpose. The pioneer works dealing with this problem
using squid giant axon have led to the idea that the cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton)
may contain some specific proteins indispensable to the excitability of the axolemma (4, 20, 24, 38). Recently, using
squid giant axons, Matsumoto and his colleagues have demonstrated that membrane excitability is destroyed by the
intraaxonal perfusion of microtubule-disrupting reagents such
tubule-associatedprotein.
© The RockefellerUniversity Press, 0021-9525/86/05/1699/11 $1.00
The Journal of Cell Biology, Volume 102, May 1986 1699-1709
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E
as colchicine, podophyllotoxin, vinblastine, and Ca ions, and
is restored by further perfusion of the microtubule polymerization buffer containing tyrosinated tubulin and a high-molecular-weight (HMW) ~ (260,000-mol-wt) protein isolated
from squid axons (7, 14-18, 27). Based on these experimental
results, a model has been proposed in which the cytoskeletal
network consisting of microtubules and 260-kD proteins plays
an important role in generating the sodium currents (13). The
following questions naturally arise: How do the microtubules
and 260-kD proteins interact with sodium channels? Are the
other cytoskeletal components such as actin filaments or other
HMW proteins involved in the membrane excitation? It seems
likely that an understanding of the molecular basis of the
above physiological findings will require further biochemical
Abbreviations used in this paper. HMW, high-molecular-weight;MAP, micro-
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biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the
subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller.
The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large
amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The a,~/-tubulin, actin, and
some other proteins were also its major constituents.
The 260-kD protein is known to play an important
role in maintaining the excitability of the axolemma
(Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91)
and was recently designated "axolinin" (Sakai, H., G.
Matsumoto, and H. Murofushi, 1985, Adv. Biophys.,
19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized
their biochemical properties. The purified axolinin
Published May 1, 1986
and morphological analyses of the molecular organization of
the subaxolemmal cytoskeleton in the squid giant axon.
Recently, Murofushi et at. (22) have partially purified the
260-kD protein, called "axolinin" by Sakai et at. (28), from
the crude extract of squid nerves and reported that the axolinin interacted with microtubules to make bundles in vitro.
Information on the in vitro nature of the other constituents
in the subaxolemmal cytoskeleton is insufficient (see reference
2), mainly because it is difficult to obtain enough fresh squid
nerves for the biochemical analysis. In the present study, we
first analyzed biochemically the molecular organization of the
subaxolemmal cytoskeleton of squid giant axons, and found
evidence that its major constituents were actin, tubulin, axolinin, and an HMW (255,000-mol-wt) protein designated here
as "255-kD protein." Secondly, axolinin and 255-kD protein
were purified in their native forms and their biochemical
properties were elucidated in vitro.
Materials and Methods
Squid Nerves
Intraaxonal Perfusion of Squid Giant Axon
After the central part of the axoplasm was squeezed out, the giant axon was
imraaxonally perfused with the following solutions successively and the perfusares were analyzed. Intraaxonal perfusion was performed according to the
method described previously (14). The giant axon was first peffused with a
solution containing 355 mM KF and 25 mM K-Hepes buffer (pH 7.3) for 10
min, then with a solution of 355 mM KI and 25 mM K-Hepes buffer (pH 7.3)
for another 10 rain, and finally with a solution of 588 mM KI, 8 mM CaC12,
and 12 mM K-Hepes buffer (pH 7.3) for the last 10 min. Perfusates were
collected for every 2.5 min in succession and analyzed by SDS PAGE using a
7.5% gel. The gel was stained with silver reagents (Bio-Rad Laboratories,
Richmond, CA).
Purification of Axolinin
Axolinin was soluble in 0.6 M NaCI but insoluble in 0.1 M NaC1 solution.
Taking advantage of this solubility, axolinin was able to be purified with high
recovery. All steps were done at 0-4"C. 50 axonal sheaths (0.3 g wet weight)
were minced with scissors and incubated for 10 rain in 1 ml of buffer A
containing 0.6 M NaCI. Buffer A was composed of 10 mM Na-MES buffer
(pH 6.8), 1 mM EGTA, 1 mM MgCI2, 0.1 mM ATP, 0.1 mM GTP, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.01 mg/ml pepstatin
A, and 0.01 mg/ml leupeptin. The sample was then centrifuged at 37,000 g for
30 rain, and the pellet was discarded. The supernatant (SI fraction; I ml),
containing - 7 mg of protein, was diluted with 5 vol of buffer A to lower the
NaCI concentration and centrifuged at 3,000 g for 5 min. The pellet (P2
fraction) was used for further steps to purify the axolinin, while the supernatant
($2 fraction) was saved for the purification of the 255-kD protein. The P2
fraction was dissolved with 1 ml of buffer A containing 0.6 M NaCI, and
centrifuged at 200,000 g for 60 rain. The supernatant ($3 fraction) was diluted
again with 5 vol of buffer A. The purified axolinin was pelleted as P4 fraction
by centrifugation at 3,000 g for 5 min.
The Journal of Cell Biology, Volume 102, 1986
The $2 fraction obtained during axolinin purification was the starting material
for the 255-kD protein purification. All steps were done at 0--4"C. 50 ml of the
$2 fraction containing 35 mg of protein was applied to a DEAE-cellulose
column (l x 13 cm) equilibrated with buffer A. The proteins were eluted with
a linear gradient created by 50 ml each of 0.1 and 0.6 M NaCI in buffer A. The
flow rate was 20 ml/h, and 5-ml fractions were collected. Each fraction eluted
from the column was analyzed by SDS PAGE. The 255-kD protein-enriched
fraction (fraction 6 in Fig. 9A) containing 4.4 mg protein was saved and further
fractionated by gel filtration through TSK-Gel G4000SW (0.75 x 60 cm). 0.5ml fractions were collected and each fraction was analyzed by SDS PAGE.
Three fractions enriched in the 255-kD protein (fractions 28-30 in Fig. 9B)
were pooled, concentrated to 0.5 ml by ultrafiltration through Diaflo PM30
membrane (Amieon Corp., Danvers, MA) and passed through Bio Gel P-10
(Bio-Rad Laboratories) column (0.8 × 10 era) equilibrated with a reassembly
buffer consisting of 0.1 M Na-MES buffer (pH 6.8), 1 mM EGTA, 0.5 mM
MgSO4, and 1 mM dithiothreitol, 0.2 mM GTP, 0.1 mM phenylmethylsulfonyl
fluoride, and 0.01 mg/ml each of pepstatin A and leupeptin.
Preparations of Microtubule Proteins, Purified
Tubulin, and Actin
Microtubule proteins were prepared by two or three cycles of temperaturedependent assembly and disassembly from squid axoplasm (27), squid optic
ganglia (6), and rat brain (29). In the case of squid axoplasm, -0.5 mg of
microtubule protein was obtained from 30 mg of axoplasmic protein which
was provided by 40 squid. To prepare rat brain microtubule proteins, 4 M
glycerol was added to the reassembly buffer during only the first assembly. The
purified tubulin was prepared from microtubule proteins obtained from rat
brain as described elsewhere (6).
Actin was prepared from rabbit skeletal muscle according to the method
developed by Spudich and Watt (30).
Co-sedimentation Experiments of Axolinin or 255-kD
Protein with Microtubules and Actin Filaments
Tubulin (2 mg/ml) or actin (1.5 mg/ml in the case of axolinin; 0.7 mg/ml in
the case of 255-kD protein) was polymerized in the presence or absence of the
purified axolinin (0.2 mg/ml) or 255-kD protein (0.1 mg/ml) by incubation for
30 min at 30"C in 0.1 ml of a solution consisting of 0.45 M Na-glutamate, 10
mM Na-MES buffer (pH 6.8), 10% dimethyl sulfoxide, 1 mM EGTA, 0.5 mM
MgCI2, 0.2 mM GTP, 0.05 mM ATP, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.01 mg/ml pepstatin A, and 0.01 mg/ml leupeptin.
The samples were then centrifuged for 30 rain at 100,000 g, and the supernatant
and pellet were analyzed by SDS PAGE.
Purification of Myosin, Spectrin, Fodrin, and FUamin
Myosin, spectrin, fodrin, and filamin were purified from rabbit skeletal muscle,
human erythrocyte, rat brain, and chicken gizzard, respectively, according to
the methods previously reported (3, 26, 35, 37).
Deoxyribonuclease I (DNase I) Column
DNase I (Sigma Chemical Co., St. Louis, MO) was covalently bound to agarose
as described by Lazarides and Lindberg (9) using CNBr-activated Sepharose 4B
(Pharmacia Fine Chemicals, Piscataway, NJ). 3 mg of DNase I was immobilized
per 1 ml of resin.
To identify axoplasmic actin, the axoplasm was analyzed using this column.
All steps were done at 4"C. Squid axoplasm (0.05 ml) was homogenized with 1
ml of a buffer B consisting of 1 mM Tris-HCl (pH 7.2), I mM EGTA, and
0.05 mM ATP. The homogenate was dialyzed against the buffer B for 24 h.
The dialysate was applied to DNase I column (0.5 × 2.5 cm). The column was
washed successively with 1 ml each of(a) buffer B, (b) 0.1 M NaCI in buffer B,
(c) 0.6 M NaCI in buffer B, (d) 0.6 M NaC1 and 2 mM CaC12 in buffer B, and
(e) 3 M guanidine-HC1. Axoplasm and each fraction eluted from the column
was analyzed by SDS PAGE.
SDS PAGE
The method developed by Laemmli (8) was used with stacking gel containing
3% polyacrylamide and with separation gel containing 3-12% linear gradient
of polyacrylamide. The gels were stained with Coomassie Brilliant Blue R-250.
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Squid, Dorytheuthis bleekeri, were collected in Sagami Bay and kept alive in a
fish preserve in Misaki Marine Biological Station, or transported and maintained in a small circular and closed-system aquarium tank in the Electrotechnical Laboratory at Tsukuba (12, 19). The nerve containing a giant axon was
dissected and rinsed with a cold solution consisting of 0.75 M glucose, 5 mM
EGTA, and 25 mM Na-MES buffer (pH 6.8). The nerve was put on a
transparent rubber, one end was cut off, and the axoplasm of the giant axon
was gently squeezed out using a rubber-coated roller. The extruded axoplasm
was collected and stored at -80"C. After extrusion, the subaxolemmal axoplasm
of giant axon, -20 um thick (see Fig. 1), remained. We called such a subaxolemmal axoplasm-enriched preparation the "axonal sheath." Axonal sheaths
were stored at -80"C until used for purification ofaxolinin and 255-kD protein.
Fin nerves containing a large number of small axons were also dissected out,
rinsed in the above solution, and stored at -80"C.
Purification of 255-kD Protein
Published May 1, 1986
Figure 2. SDS PAGE of the extruded
Immunological Methods
axoplasm of the squid giant axon and
the axonal sheath extract. (Lane 1)
Standard proteins used as mobility
markers consisting of phosphorylase b
(92 kD), bovine serum albumin (68 kD),
ovalbumin (45 kD), carbonic anhydrase
(31 kD), and cytochrome c (12 kD);
(lane 2) axoplasm extruded from squid
giant axon by a roller; (lane 3) axonal
sheath proteins extracted with 3 vol of
0.6 M NaC1, 10 mM CaC12,and 10 mM
Na-Hepes buffer (pH 7.2) for 10 min at
0*C; (lane 4) axonal sheath proteins extracted with 3 vol of 8 M urea, 2% SDS,
5% 2-mercaptoethanol, and 0.1 M TrisHCI buffer (pH 6.8) for 2 min at 100*C.
Note that, among HMW polypeptides
(A, 320 kD; B, 260 kD; C, 255 kD), the
axolinin (B) and 255-kD protein (C) are
enriched in the axonal sheath extract,
while the 320-kD protein (A) is detected
only in the extruded axoplasm. Tubulin
(D and E) and actin (F) are major constituents of both the central and subaxolemmal axoplasm.
Antisera to the purified axolinin were elicited in rabbits. Approximately 0.2 mg
of antigen in 0.5 ml of 0.6 M NaC1 and l0 mM Na-MES buffer (pH 6.8) was
emulsified with an equal volume of complete Freund's adjuvant, and intracutaneously injected at multiple sites along the backs of two rabbits. The second
and third injections were repeated 4 and 5 wk after the first injection, respectively. The rabbits were bled a week after the third injection. The IgG fraction
was further purified from the antisera using DEAE-cellulose ion-exchange
column chromatography.
Immunoblotting was performed by SDS PAGE followed by electrophoretic
transfer to nitrocellulose sheets as described by Vaessen et al. (36). Nitrocellulose
sheets were treated with the anti-axolinin antibody followed by horseradish
peroxidase-conjugated antibodies (Miles Laboratories, Inc., Elkhart, IN). The
sheets were then treated with 4-chloro-l-naphtol solution to localize the peroxidase.
Protein Assay
Protein concentrations were determined according to the protocol of Lowry et
al. (11), using bovine serum albumin as a standard. For rapid assay of protein
concentrations of column elutes, the Bradford procedure (1) was used with
tubulin as a standard.
Low-angle Rotary-shadowing Electron Microscopy
been squeezed out followed by intraaxonal perfusion. T h e giant axon
is s u r r o u n d e d by the bundle o f small a x o n s (B). After the intraaxonal
perfusion, the s u b a x o l e m m a l axoplasm (.), - 2 0 ~ m thick, remains
u n d e r the a x o l e m m a (AL).
remained under the axolemma (Fig. I). This subaxolemmal
axoplasm--enriched preparation was called the "axonal
sheath." First, we compared the protein composition of the
axonal sheath with that of the extruded axoplasm by the use
of SDS PAGE (Fig. 2). We faced a little difficulty in the
analysis of the axonal sheath proteins; some connective tissues
were by no means solubilized even with a solution containing
8 M urea, 2% SDS, and 5% 2-mercaptoethanol, and disturbed
the electrophoreogram (see Fig. 2, lane 4). To avoid this
difficulty, a solution containing 0.6 M NaCI, 10 mM CaCI2,
and 10 mM Na-MES buffer (pH 6.8) was used according to
the method of Sakai and Matsumoto (27). This solution
solubilized almost the same proteins as the above urea-SDS
solution without any disturbance of the electrophoresis system
(compare lane 3 in Fig. 2 with lane 4). As a result, it became
clear that the axonal sheath was rather simple in its protein
composition when compared to the central axoplasm. The
electrophoretic banding pattern of the axonal sheath was
characterized by large amounts of the two HMW polypeptides
designated 255 kD and 260 kD. Judging from its molecular
mass, the 260-kD polypeptide was considered to be identical
to the "axolinin" designated by Sakai et al. (28). The 255-kD
protein and axolinin accounted for 1.7 and 1.5% of the total
protein in extruded axoplasm of a giant axon, and 5.2 and
8.9% of that in the axonal sheath extract, respectively. Considering that the protein composition of the whole axonal
sheath solubilized with a solution containing 8 M urea and
2% SDS highly resembles that of the axonal sheath extract
(see Fig. 2), we can conclude that both 255-kD protein and
axolinin were highly concentrated in the subaxolemmal axoplasm. In addition to these proteins, the 45-kD, 53-kD, and
55-kD polypeptides were also predominant in the extracts of
axonal sheaths, which might correspond to actin, fl-tubulin,
and a-tubulin, respectively.
To demonstrate that axolinin, 255-kD protein, tubulin, and
actin were really located in the subaxolemmal axoplasm, a
Kobayashi et al. Subaxolemmal Cytoskeleton in SquidGiant Axon
1701
Results
Protein Composition of Subaxolemmal Cytoskeleton
in Squid Giant Axon
After the central part of the axoplasm of a squid giant axon
was squeezed out, the peripheral axoplasm, ~20 zm thick,
Figure 1. Light micrograph o f transverse section o f the squid nerve
containing a giant axon (GA) after the central part o f axoplasm has
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To study the morphology of the isolated axolinin and 255-kD protein molecules, we used the low-angle rotary-shadowing technique, mainly according to
the method developed by Tyler and Branton (34). 200 ~1 of a solution containing axolinin (0.025 mg/ml), 0.6 M NaC1, 50% glycerol, and 10 mM Na-MES
buffer (pH 6.8) or a solution containing 255-kD protein (0.035 mg/ml), 0.1 M
NaCI, 50% glycerol, and 10 mM Na-MES buffer (pH 6,8) was sprayed onto
freshly cleaved mica. The droplets on the mica were then dried at room
temperature under vacuum (1 × 104 Tort) in an Eiko freeze-etch device, FD2 (Eiko Engineering, Mito, Japan), for 10 rain. Platinum was then rotaryshadowed at an angle of 5* followed by a coating from above with carbon. The
replicas were floated off on the distilled water and picked up on the formvarfilmed grids. They were examined in a Hitachi 1 I-DS electron microscope at
an accelerating voltage of 75 kV. Electron microscope negatives were contactreversed and printed as negative images.
Published May 1, 1986
Figure 3. SDS PAGE of the proteins released from the interior of a
Figure4. SDS PAGE of microtubule proteins prepared from rat brain
and squid axoplasm. (A) Electrophoretic banding pattern of isolated
microtubules. The HMW-MAPs of squid axoplasmic microtubules
(lane 4) is rather simple, consisting of axolinin (260K)and a 320-kD
protein (320K),while the rat brain microtubules (lane I) contain four
polypeptides ranging from 300 to 350 kD as HMW-MAPs. Lanes 2
and 3 show axoplasm of squid giant axon. (B) Heat treatment of
microtubule proteins at 100*C for 2 rain. In rat brain microtubule
preparations, 320-kD and 350-kD MAPs are precipitated together
with tubulin after heat treatment followed by centrifugation (lane 2),
while 300-kD and 310-kD MAPs are heat resistant and recovered in
supernatant (lane 1). In the case of squid axoplasmic microtubules,
both axolinin and 255-kD protein were precipitated by heat treatment
(lane 4), and no MAPs are recovered in supernatant (lane 3).
squid giant axon was intraaxonally perfused with several kinds
of solutions after its central part of the axoplasm was squeezed
out, and the perfusate was collected and analyzed by the use
of SDS PAGE combined with the silver staining method (Fig.
3). As shown in lanes 3-6 of Fig. 3, appreciable amounts of
axolinin and 255-kD protein were released together with other
proteins containing tubulin by the perfusion of a solution
consisting of 355 m M KF and 25 mM K-Hepes buffer (pH
7.3). This solution was routinely used as the internal solution
in physiological experiments to maintain the excitability of
the axolemma. After the perfusion solution was switched to
the solution composed of 355 m M KI and 25 mM K-Hepes
buffer (pH 7.3), large amounts of axonal proteins containing
tubulin, actin, and axolinin were released (Fig. 3, lanes 7-10).
It should be noted that after the KI solution, a great deal of
actin came off together with two H M W polypeptides (235and 240-kD) which might correspond to the polypeptides
called "fodrin" by Morris and Lasek (21). This seemed to
indicate that actin was located very close to the axolemma or
that actin filament was stabilized inside axons compared to
microtubules. Almost no polypeptides were detected in the
perfusate obtained by perfusion with a solution composed of
355 mM KI, 8 mM CaCl2, and 25 mM K-Hepes buffer (pH
7.3).
These findings have led us to conclude that the subaxolemmal axoplasm was mainly composed of axolinin (8.9% of
total protein), 255-kD protein (5.2%), a,~-tubulin (8.0%),
actin (5.4%), and some other polypeptides. Next, we have
Microtubule proteins from the extruded axoplasm of squid
giant axons were prepared by two cycles of temepraturedependent assembly and disassembly as described by Sakai
and Matsumoto (27). Approximately 0.5 mg of microtubule
proteins was obtained from 30 mg of axoplasmic proteins
which was provided by 40 squid. Microtubules prepared from
squid axoplasm contained two main components of H M W
microtubule-associated proteins (HMW-MAPs) of 320 kD
and 260 kD (Fig. 4A, lane 4), both of which were precipitated
by heat treatment for 2 rain at 100*C (Fig. 4B, lane 4). The
260-kD MAP was considered to be axolinin, As shown in Fig.
2, the 320-kD MAP was localized only in the central axoplasm
but not in the subaxolemmal axoplasm. For comparison,
microtubules were prepared from rat brain using the same
isolation procedure. The rat brain microtubules were shown
to contain four polypeptides ranging from 300 to 350 kD as
HMW-MAPs (Fig. 4A, lane I). The 300- and 310-kD MAPs
were heat resistant (Fig. 4B, lane 1), while the 320- and 350kD MAPs were heat sensitive (Fig. 4/~', lane 2). Although it is
difficult to simply compare the HMW-MAPs of squid giant
The Journal of Cell Biology,Volume 102, 1986
1702
studied some biochemical properties of these proteins isolated
from the squid giant axon.
Isolation of Microtubule Proteins from Axoplasm of
Squid Giant Axon
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squid giant axon during intraaxonal perfusion, first with the KF
solution for l0 rain, then with the KI solution for another 10 rain,
and finally with the KI-Ca solution for the last l0 min (see Materials
and Methods for details). Perfusates were collected for every 2.5 min
in succession and analyzed by SDS PAGE combined with silver
staining method. (Lane 1) Microtubule proteins prepared from squid
optic ganglia; (lane 2) mixture of the purified axolinin and 255-kD
protein; (lanes 3-6) perfusates of KF solution; (lanes 7-10) perfusates
of KI solution; (lanes 11-13)perfusates of KI-Ca solution. It is clearly
shown that the KF solution releases large amounts of axolinin (260K)
and 255-kD protein (255K) together with tubulin (7) and actin (A).
Note that an appreciable amount of actin is released together with
"fodrin" (arrow) after the perfusion with KI solution.
Published May 1, 1986
Figure6. Purification of axo-
Figure5. Identification of actin in the axoplasm of squid giant axon
axon with those of rat brain, it is safe to say that the H M W MAP family of squid giant axon is rather simple, and that the
central and peripheral parts of the axoplasm contain different
types of HMW-MAPs, the 320-kD MAP and axolinin, respectively.
Identification o f Actin in Axoplasm o f Squid
Giant Axon
To study whether the 45-kD polypeptide in the axoplasm was
really actin, the extruded axoplasm was analyzed using the
DNase I affinity column according to the method developed
by Lazarides and Lindberg (9) (Fig. 5). As a result, the 45-10)
polypeptide was specifically adsorbed on and eluted from the
DNase I column under the same condition as actin, indicating
that the 45-kD polypeptide was actin. Actin was identified in
large amounts in both central and peripheral axoplasm (see
Fig. 2). In the condition used here, no actin-binding protein
was trapped by the DNase I column.
fraction; Fig. 6, lane 3). The P2 fraction was dissolved in 0.6
M NaCl solution, and the precipitation-centrifugation procedure was repeated two more times. As a result, the axolinin
was obtained in a high degree of purity (Fig. 6, lane 8).
Approximately 1 mg of the purified axolinin was obtained
from 50 giant axonal sheaths. Fin nerves were also a good
source to purify the axolinin (22). Approximately 8 mg of the
axolinin was purified from 50 fin nerves (l g wet weight).
Molecular Shape o f Axolinin Molecule
The molecular shape of purified axolinin molecules in 0.6 M
NaC1 solution was analyzed by means of low-angle rotaryshadowing electron microscopy. The axolinin molecule
looked like a straight rod ~105 nm long with a globular head
at one end (Fig. 7). This molecule tended to form a ring
structure by head-to-tail association within one molecule (Fig.
7, a, e, and f ) . Occasionally, some molecules were split into
two strands which were arranged parallel to each other (Fig.
7d), suggesting that the native axolinin might be a homodimer in 0.6 M NaCl solution.
Co-sedimentation Experiments o f ,4xolinin with
Microtubules and,4ctin Filaments
We found that axolinin was soluble in 0.6 M NaCI solution
but insoluble in 0.1 M NaCI solution. Because of this characteristic, only successive centrifugations separated axolinin
from other proteins with high recovery (Fig. 6). In the crude
extract (SI fraction; see Materials and Methods for details)
containing 0.6 M NaCI from axonal sheaths, axolinin, 255kD protein, tubulin, actin, and some other proteins including
neurofilament 60-kD protein were prominent (Fig. 6, lane 2).
When the extract was diluted to 0.1 M NaC1 solution followed
by centrifugation, in the pellet (P2 fraction) the axolinin was
strikingly enriched (Fig. 6, lane 4) and the 255-kD proteins
and other proteins were recovered in the supernatant ($2
Binding ability of the purified axolinin to microtubules and
actin filaments was studied by co-sedimentation experiments
(Fig. 8). After the incubation of tubulin or actin with the
purified axolinin at 30"C for 30 min, the samples were centrifuged to precipitate assembled microtubules or actin filaments. The proteins in the supernatant and pellet were then
analyzed by electrophoresis. Lacking either tubulin or actin,
about 16% of the total amount of axolinin was sedimented.
However, in the presence of tubulin, the sedimented axolinin
remarkably increased in amount, to 70% of the total axolinin.
On the other hand, actin filaments did not induce the sedimentation 'of axolinin. Considering that the axolinin itself
had no effects on the sedimentation properties of tubulin and
actin, it was concluded that axolinin could bind to microtu-
Kobayashiet al. SubaxolemmalCytoskeletoninSquidGiantAxon
1703
Purification o f Axolinin from Axonal Sheaths
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by the use of DNase I column. (Lanes 1 and 7) Extract of axoplasm
applied to DNase I column (see Materials and Methods for details);
(lane 2) combined fraction of flow-through and low salt wash; (lane
3) 0.1 M NaC! eluate; (lane 4) 0.6 M NaCI eluate; (lane 5) 0.6 M
NaCI-2 mM CaCI2 eluate; (lane 6) 3 M guanidine-HC1 eluate. The
45-kD protein is specifically adsorbed on and eluted from the column
(lane 6), indicating that this 45-kD protein is actin.
linin. The protein composition of each fraction in the
steps of the purification was
analyzed by SDS PAGE. For
details of the purification procedure, see Materials and
Methods. (Lane 1) Axoplasm;
(lane 2) S l fraction, 0.6 M
NaC1 extract of axonal
sheath; (lane 3) $2 fraction,
0.1 M NaCl-soluble fraction
from S1; (lane 4) P2 fraction,
0.1 M NaCl-insoluble fraction from S l; (lane 5) $3 fraction, supematant after centrifugation of P2 dissolved in
0.6 M NaCl; (lane 6) P3 fraction; (lane 7) $4 fraction, 0.1
M NaCl-soluble fraction
from $3; (lane 8) P4 fraction,
0.1 M NaCl-insoluble fraction from $3. The axolinin (260K) is purified with high recovery as
P4 fraction (lane 8). Note that the 255-kD protein is enriched in $2
fraction (lane 3).
Published May 1, 1986
structures (a, e, and f).
rofushi et al. using different conditions for the co-sedimentation experiments (22).
Purification of 255-kD Protein from Axonal Sheaths
Figure 8. Co-sedimentation experiments on axolinin with microtubules and actin filaments. For details of experimental conditions, see
Materials and Methods. Lanes 1, 3, 5, 7, and 9 represent the protein
composition of the supernatant of each experiment, and lanes 2, 4,
6, 8, and I0 represent that of the pellet. (Lanes 1 and 2) Only tubulin
was incubated; (lanes 3 and 4) axolinin and tubulin were incubated;
(lanes 5 and 6) only axolinin was incubated; (lanes 7 and 8) only
actin was incubated; (lanes 9 and 10) axolinin and actin were incubated. Axolinin specifically co-sediments with microtubules.
bules but not to actin filaments in vitro. Addition of 2 mM
CaC12 to the incubation medium had no effect on the cosedimentation of the axolinin with actin filaments (data not
shown). Recently, similar results have been obtained by Mu-
The Journal of Cell Biology,Volume 102, 1986
The $2 fraction obtained during the purification of the axolinin was the starting material for the preparation of the 255kD protein. Upon SDS PAGE of the $2 fraction, the 255-kD
protein, a,/~-tubulin, and actin were prominent, and some
other polypeptides such as neurofilament 60-kD protein were
also present (see Fig. 6, lane 3). DEAE-cellulose column
chromatography yielded a fraction enriched in the 255-kD
protein (Fig. 9A). A fraction of fraction 6 shown in Fig. 9A
was further fractionated by gel filtration (Fig. 9 B). As a result,
~0.7 mg of the highly purified 255-kD protein was obtained
from 50 axonal sheaths. In sharp contrast with axolinin, the
purified 255-kD protein was soluble in both 0.1 and 0.6 M
NaCI solution. When the purified 255-kD protein was coelectrophoresed with the purified axolinin, it was evident that
these proteins were distinct in their molecular masses (Fig.
9C).
Molecular Shape of 255-kD Protein Molecule
In low-angle rotary-shadowing electron microscopic images,
the 255-kD protein molecule appeared as a characteristic
horseshoe-shaped structure with diameter of ~35 nm (Fig.
10). Neither in 0.1 nor in 0.6 M NaCI solution was there an
indication that this protein formed aggregates.
Interaction o f the 2 5 5 - k D Protein with Microtubules
and Actin Filaments
The microtubules isolated from the squid giant axon did not
contain the 255-kD protein (see Fig. 4), indicating that the
1704
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Figure 7. Morphology of axolinin molecule in 0.6 M NaCI solution in rotary-shadowed preparations. Axolinin molecule appears as a straight
rod about 105 nm long with a globular head at one end (a-c). Some molecules are split into two thinner strands (a and d) and form ring
Published May 1, 1986
Figure 9. Purification of the 255-kD protein.
(A) Fractionation by DEAE-cellulose column
chromatography. The $2 fraction in Fig. 6
was applied on the column. Fraction No. 6
was further fractionated. (B) Fractionation
by gel filtration through TSK-Gel G4000SW.
Three fractions, fractions 28-30, were combined. (C) Comparison of the molecular mass
of the purified axolinin (lane 2) and the 255kD protein (lane 3). When these proteins are
co-electrophoresed (lane 4), two bands are
dearly separated. Lanes 1 and 5 show the
protein composition of axoplasm extruded
from giant axon.
Downloaded from on June 15, 2017
Figure 10. Molecular shape of the 255-kD protein molecule in 0.1 M NaCI solution. The 255-kD protein molecule appeared as a characteristic
horseshoe-shaped structure -30-35 nm in diameter.
255-kD protein might not bind to microtubules. Actually,
when the purified 255-kD protein was incubated with microtubules followed by centrifugation, only 8% o f the total
amount of the 255-kD protein was sedimented. The same
amount of 255-kD protein was precipitated in the absence of
tubulin (Fig. 11). On the other hand, in the presence o f actin
filaments, 62% o f the total 255-kD protein was sedimented.
The presence of the 255-kD protein had no effect on the
sedimentation properties oftubulin and actin. Taken together,
it was concluded that the 255-kD protein was actin binding
but not tubulin binding.
Interaction of the 255-kD protein with actin was further
studied by viscometry at a low-shear rate (Fig. 12). Solution
containing 255-kD protein and G-actin was sucked up into a
Kobayashiet al. Subaxolemrnal Cytoskeleton in Squid Giant Axon
1705
Published May 1, 1986
Figure 11. Co-sedimentationexperiments of the 255-kD protein with
creased gradually, but compared to the chicken gizzard filamin its ability to gel the actin solution was very low.
Immunological Reactivity of Anti-Axolinin Antibodies
with Squid Axoplasmic Proteins and Some Other
H M W Proteins
gel
~800
.>
>
E
400
<
j
0
several HMW proteins. (A) SDS polyacrylamide gels stained with
Coomassie Brilliant Blue; (B) nitrocellulosereplica stained with antiaxolinin. (Lane 1) Rabbit skeletal muscle myosin; (lane 2) purified
axolinin; (lane 3) chicken gizzard filamin; (lane 4) human erythrocyte
spectrin; (lane 5) rat brain fodrin; (lane 6) squid axoplasm; (lane 7)
purified 255-kD protein. This antibody exclusivelyreacts with axolinin.
fro
0.05
i.
01
Protein (mg/ml)
Figure 12. Interaction of the 255-kD protein (I), axolinin (0), or
chicken gizzard filamin (A) with actin filaments as studied by lowshear viscometry. The basic mixture consisted of 0.45 M Na-glutamate, 10 mM Na-MES buffer (pH 6.8), 1 mM EGTA, 0.5 mM
MgCi2, 0.1 mM ATP, and 0.2 mg/ml actin. Samples were incubated
for 30 rain at 37"C before measurement of viscometry. The viscosity
of actin solution increases gradually with increasing concentration of
the 255-kD protein, but its ability to gel the actin solution is very low
compared to that of filamin. Axolinin does not increase the viscosity
of the actin solution.
To clarify the difference or similarity of axolinin with other
HMW proteins, especially with the 255-kD protein, antisera
to the purified axolinin were elicited in rabbits and the immunological properties of the axolinin were characterized by
the immune blotting procedure using peroxidase (Fig. 13).
When the squid axoplasm was analyzed by SDS PAGE followed by immune blotting, anti-axolinin stained exclusively
the band of axolinin, but not the band of 255-kD protein
(Fig. 13, lane 6). The purified 255-kD protein showed no
cross-reactivity with the anti-axolinin (Fig. 13, lane 7), while
this antibody strongly stained the band of the purified axolinin
(Fig. 13, lane 2). Since the axolinin molecule resembled a
straight rod (see Fig. 7), the following HMW proteins showing
rod-like molecular shape were purified and their reactivity to
the anti-axolinin was analyzed: rabbit skeletal muscle myosin,
chicken gizzard fdamin, human erythrocyte spectrin, and rat
brain fodrin. None of them showed any cross-reactivity (Fig.
13, lanes 1, 3, 4, and 5). Furthermore, this antibody did not
stain any of the HMW-MAPs obtained from rat brain (data
not shown).
Gel Filtration Profile o f Axolinin and 255-kD Protein
capillary pipette, and the falling ball assay was performed after
30 min at 37"C. As a result, with increasing concentration of
the 255-kD protein, the viscosity of the actin solution in-
To estimate the subunit configuration of the axolinin and the
255-kD protein, both proteins were passed through a TSKgel G4000SW column together with rat brain spectrin (tetramer), rabbit skeletal muscle myosin (dimer), and chicken
gizzard fdamin (dimer) (Fig. 14). The axolinin in the 0.6-M
The Journal of Cell Biology,Volume 102, 1986
1706
Downloaded from on June 15, 2017
microtubules and actin filaments. For details of the experimental
conditions, see Materials and Methods. Lanes 1, 3, 5, 7, and 9
represent the protein composition of the supernatant, and lanes 2, 4,
6, 8, and 10 represent that of the pellet. (Lanes 1 and 2) 255-kD
protein and tubulin were incubated; (lanes 3 and 4) only tubulin was
incubated; (lanes 5 and 6) only 255-kD protein was incubated; (lanes
7 and 8) 255-kD protein and actin were incubated; (lanes 9 and 10)
only actin was incubated. 255-kD protein co-sediments with actin
filaments, but not with microtubules.
Figure 13. Immunological reactivity of anti-axolinin antibodies with
Published May 1, 1986
A
E
tO
oo
¢,4
¢)
tt~
.Q
o
e~
<
10
15
20
25
Elution Volume (ml)
the same column under the same condition. The peak positions of these proteins are indicated by A, B, and C, respectively. The 255-kD
protein was shown to elute at the position indicated by arrowheads.., flow-through fractions.
NaCI solution was eluted between myosin and filamin. Considering that myosin and filamin molecules as well as axolinin
molecules appear as rod-shaped structures ~ 100-150 nm in
length, it is safe to say that the axolinin molecule in the 0.6M NaC1 solution may be a dimeric form rather than a
monomeric form. The 255-kD protein was eluted between
axolinin and filamin. Taking into consideration that 255-kD
protein molecule appears as a horseshoe-shaped structure with
diameter of 35 nm and that a globular protein is eluted more
slowly than rod-shaped proteins with similar molecular
masses, it may well be that the purified 255-kD protein is also
a dimeric form rather than a monomeric form.
Our present results have clearly demonstrated that large
amounts of two distinct unique HMW proteins, axolinin and
255-kD protein, are localized in the subaxolemmal cytoskeleton of squid giant axon together with tubulin and actin, and
that the purified axolinin and 255-kD protein can bind to
microtubules and actin filaments in vitro, respectively. These
HMW proteins were also found in the central axoplasm
extruded from squid giant axons, but only in small amounts.
We suppose that these two proteins, together with microtubules and actin filaments, may form a densely packed cytoskeletal network tightly bound to the axolemma, since this
subaxolemmal cytoskeleton is hardly extruded when the axon
is squeezed with a roller (see Fig. 1). The morphological
aspects of the subaxolemmal cytoskeleton will be described
in the following paper (33).
When the microtubule proteins were prepared from the
axoplasm of squid giant axons, the axolinin was co-purified
with microtubules. In this sense, the axolinin can be categorized as HMW-MAPs. Actually, in vitro, the purified axolinin
was co-sedimented with microtubules. Recently, Murofushi
et al. have shown that the axolinin has no ability to promote
the polymerization of purified tubulin, while MAP-2 prepared
from porcine brain has this ability (22). Furthermore, the
molecular shape of the axolinin molecule demonstrated in
this study seems distinct from that of the MAP-2 molecule,
which has been shown to look like a very thin flexible strand
~ 180 nm in length without a head-like globular structure on
its end. The axolinin was heat sensitive, while MAP-2 was
heat resistant. When these characteristics are taken together,
it can be concluded that axolinin is not a MAP-2-1ike protein.
Since our knowledge of the in vitro nature of the mammalian
HMW-MAPs other than MAP-2 is still fragmentary, it is
premature to further compare axolinin with the mammalian
HMW-MAPs. However, it is interesting to speculate that
some of the mammalian HMW-MAPs may resemble axolinin
and constitute the subaxolemmal axoplasm in mammalian
axons.
The 255-kD protein was also enriched in the subaxolemmal
cytoskeleton of squid giant axon. Judging from its ability to
bind to actin filaments, its molecular shape, and its solubility
in 0.1 M NaC1 solution, it can be said that the 255-kD protein
is not a degradation product of the axolinin. This was confirmed by the fact that the 255-kD protein showed no crossreactivity with anti-axolinin antibodies. The localization of
the 255-kD protein in the squid giant axon, its binding
property to actin filament, and its molecular mass of 255 kD
have persuaded us to consider the relation of the 255-kD
protein and spectrin-like proteins. Recently, a spectrin-like
Kobayashi et al. Subaxolemmal Cytoskeleton in Squid Giant Axon
1707
Discussion
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Figure 14. Gel filtration profile of purified axolinin. The purified axolinin in buffer A containing 0.6 M NaC1 (see Materials and Methods) was
passed through a TSK-gel G4000SW column equilibrated with buffer A containing 0.6 M NaCI. Absorbance at 280 nm of each fraction was
monitored. For comparison, rat brain fodrin (tetramer), rabbit muscle myosin (dimer), and chicken gizzard filamin (dimer) were passed through
Published May 1, 1986
protein, fodrin/calspectin, has been isolated from mammalian
brain (3, 5, 32), and revealed to be localized just beneath the
axolemma in mammalian myelinated axons (3, I0, 25). Rat
fodrin was reported to be composed of two distinct HMW
polypeptides (designated 235 kD and 240 kD) and to bind to
actin filaments. However, the 255-kD protein should not be
regarded as a spectrin-like protein, mainly for the following
reasons. (a) The molecular shape of the 255-kD protein
resembled a horseshoe-shaped globular structure ~35 nm in
diameter, while the fodrin molecule looked like a flexible rod
~100 nm in length in a dimeric form. (b) Antibodies to
chicken erythrocyte spectrin cross-reacted with rat brain fodrin, but not with the squid 255-kD protein (data not shown).
(c) As Morris and Lasek have pointed out (21), two HMW
polypeptides (235 kD and 240 kD) are present in the same
amounts in the axoplasm of the squid giant axon, and their
molecular masses are completely identical to the mammalian
fodrin (for example, see Fig. 3). Therefore, at present, it is
difficult to identify the HMW proteins in vertebrate cells that
resemble the axolinin or the 255-kD protein. We believe that
this difficulty does not represent a species difference and that
the proteins similar to axolinin and 255-kD protein will be
identified in vertebrate axons in the future, since at least
axolinin is known to play an important role in the excitation
of the axolemma in squid giant axon (14).
It remains to be elucidated whether the 255-kD protein and
axolinin can bind to neurofilaments, but so far there is no
data which suggests that these proteins are neurofilamentbinding proteins. Pant et al. (23) analyzed the protein composition of the neurofilament-enriched fraction obtained from
squid giant axons by SDS PAGE. Judging from their data, it
is clear that only a trace of 260 kD and 255 kD proteins are
co-purified with neurofilaments. Furthermore, a recent study
by Zackroff and Goldman (39) has also shown that the
neurofilaments purifed from squid brain contain the 220-kD
protein but neither the 260-kD nor the 255-kD proteins.
Tasaki and his colleagues have pointed out the importance
of the protein layer beneath the axolemma of a squid giant
axon as a reservoir of a specific protein indispensable to the
electrical excitability of the axolemma (31). They have stressed
that this electrical excitability deteriorates when a certain
specific subaxolemmal protein is released from this protein
layer. One of such proteins has been identified as a 12-kD
protein. It may be, however, that this 12-kD protein is a
component of the intraaxonally perfused pronase itself or a
proteolytic product derived from the subaxolemmal proteins,
as Tasaki et al. have stated themselves (31). It should be noted
that the amount of the 12-kD polypeptide is not great either
in the extruded axoplasm or in the axonal sheath. On the
other hand, Matsumoto and his colleagues have suggested
that axonal microtubules with the 260-kD protein (axolinin)
may take a direct role in generating sodium currents in squid
giant axon (7, 14-18, 27). Thus, it is possible to speculate that
the 12-kD protein may be a degradation product of tubulin
or axolinin. These studies have provided the crucial information on the physiological roles of the subaxolemmal cytoskeleton in membrane excitation, and the present study has
clearly demonstrated the molecular organization of the subaxolemmal cytoskeleton in squid giant axon. However, the most
important and fundamental problem remains to be elucidated: what kind of interaction occurs between sodium chan-
1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72:248-254.
2. Gainer, H., and V. S. Gainer. 1976. Proteins in the squid giant axon. In
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has a morphology related to spectrin. Cell 28:843-854.
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5. Kakiuchi, S., K. Sobue, K. Kanda, K. Morimoto, S. Tsukita, S. Tsukita,
H. lshikawa, and M. Kurokawa. 1982. Correlative biochemical and morphological studies of brain calspectin: a spectrin-like calmodulin-binding protein.
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6. Kobayashi, T. 1982. Fractionation and electrophoretic analysis of microtubule proteins from rat brain. In Biological Functions of Microtubules and
Related Structures. H. Sakai, H. Mohri, and G. G. Borisy, editors. Academic
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squid nerve fully retains C-terminal tyrosine. J. Biochem. 92:647-652.
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9. Lazarides, E., and U. Lindberg. 1974. Actin is the naturally occurring
inhibitor of deoxyribonuclease I. Proc. Natl. Acad. Sci. USA. 71:4742-4746.
10. Levine, J., and M. Willard. 1981. Fodrin:axonally-transported polypeptides associated with the internal periphery of cells. Z Cell Biol. 90:631-643.
11. Lowry, O. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951.
Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265275.
12. Matsumoto, G. 1976. Transportation and maintenance of adult squid
(Doryteuthis bleekeri) for physiological studies. Biol. Bull. 150:279-285.
13. Matsumoto, G. 1983. A proposed membrane model for generation of
sodium currents in squid giant axons. J. Theor. Biol. 107:649-666.
14, Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai.
1983. Axonal microtubules necessary for generation of sodium current in squid
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77:77-91.
15. Matsumoto, G., T. Kobayashi, and H. Sakai. 1979. Restoration of the
excitability of squid giant axon by tubulin-tyrosine ligase and microtubule
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16. Matsumoto, G., H. Murofushi, and H. Sakai. 1980. The effects of
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18. Matsumoto, G., and H. Sakai. 1979. Restoration of membrane excita-
The Journal of Cell Biology, Volume 102, 1986
1708
nels and subaxolemmal cytoskeletons at the molecular level
during membrane excitation? There seem to be far more
axolinin and 255-kD protein molecules than sodium channels
in a squid giant axon. Taken together with the morphological
data shown in the following paper (33), we can conclude that
neither axolinin nor 255-kD protein works as a cross-linker
between sodium channels and the subaxolemmal cytoskeleton. We have just begun to search for the cytoskeletal proteins which can directly interact with sodium channels. For
this purpose, we believe the squid giant axon with its simple
protein composition of the subaxolemmal cytoskeleton will
continue to serve as an ideal model system.
We wish to t h a n k Misaki Marine Biological Station for allowing us
use of its facilities. Thanks are also due to Dr. M. Ichikawa for
collecting the squid nerves.
This study was supported in part by research grants from the
Ministry of Education, Science and Culture, Japan, and from the
Mitsubishi Foundation.
Received for publication 22 July 1985, and in revised form 20
November 1985.
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Kobayashi et al. Subaxolemmal Cytoskeleton in Squid Giant Axon
1709